(1) Field of the Invention
The present invention relates to novel antiviral peptides and preparations. More particularly, the invention relates to antiviral peptides derived from venom alpha-neurotoxins, and are capable of inhibiting viral neuraminidases.
(2) Description of Related Art
There are several sources of toxins that can be harmful to both humans and animals, such as radiation, inorganic and organic substances. Neurotoxins are toxins which specifically affect neurons through interaction with membranes and ion channels. Most animal venoms can be classified as neurotoxins, such as those found in bees, snakes, scorpions, and spiders. Detoxification of such neurotoxins is a goal when treating those affected. Also, antiviral solutions can be derived from neurotoxins.
Sanders, M., et al., (Ann. N.Y. Acad. Sci. (1953) 58: 1-12) discloses multiple species of snake venoms detoxified by chemical oxidation. An antiviral test system measured effects of the venom derivatives on survival rates of rats, mice, and monkeys experimentally infected with polio viruses. The choice of chemical reactions was of prime importance. Oxidative detoxification of one class of whole venoms produced antiviral activities. Detoxifications by reaction with formaldehyde produced inert materials. The significance of the differences in reactivity was two-fold. First, the formaldehyde-treated group served as an excellent control for the active, oxidized venoms because the test animals in the two groups received the same quantities of venom, thus negating nonspecific protein interactions as the cause of the difference in activity. Second, if the active ingredients in the venoms were proteins, a fact not known at the time, the chemical groups modified by the formaldehyde could be implicated in the antiviral effect. Of prime importance was restriction of the antiviral activities to oxidatively detoxified venoms that contained alpha-neurotoxins. Those venoms included all species of cobras tested, and the Banded Krait and South American rattlesnake, Crotalus duressus terrificus. 
Twenty two years later one of the present inventors proved, through separations technology, that the specific precursors of the antiviral activities in neurotoxic venoms were the alpha-neurotoxins (Miller, et al., Biochim. Biophys. Acta (1975) 496: 192-196). The alpha-neurotoxins from two of the venoms, the Thailand cobra and the Banded Krait, were isolated in chemically pure form and detoxified by the identical oxidation reaction employed in the original work by Sanders. Antiviral activities of those materials were quantitated by reductions of plaque counts formed by the Semliki Forest Virus (a neurotropic virus like the polio virus) on cultured sheets of baby hamster kidney (BHK) cells. Detoxification by reduction and alkylation of the toxins produced the same nontoxic antiviral peptide actions. (Miller, K. D., in “Workshop on Modified Neurotoxin: Treatment of Amyotrophic Lateral Sclerosis”, Bureau of Biologicals, NINCDS, Apr. 5, 1979: p 63-76). Investigations continued with evaluations of those purified antiviral polypeptides in several tissue culture and animal test systems employing the herpes simplex virus (Yourist, J. E., et al., J. Gen. Virol. (1983) 64: 1475-1481). Further, Yourist demonstrated inhibition of several protein kinases, including a protein kinase derived from human myelin, by both the oxidized and reduced and alkylated peptides (Yourist, J E, Doctoral dissertation, University of Miami, 1979).
Important to an understanding of the present invention are the similarities in chemical structures among a wide variety of animal alpha-neurotoxins. Those similarities include closely related amino acid sequences in key parts of the toxin molecules (Yang, C. C., Toxicon, (1974) 12: 1-43). A rigid tertiary conformation of the conserved region after detoxification by disulfide bond scission is suggested by the triple-stranded beta-pleated sheet structure of the native toxin as reported by Walkinshaw (Proc. Nat. Acad. Sci. (1980) 77: 2400-2404).
Antiviral peptides derived from alpha-neurotoxins are formed by scission of the toxin disulfide bonds with resultant conformational changes and elimination of toxicity. Use of strong oxidants for this purpose is problematic because this may result in modifications of other amino acids in these peptides, especially tyrosine, histidine, tryptophane and lysine, common constituents of the alpha-neurotoxins (Yang, C. C., Toxicon (1974) 12: 1-43; Elliott, K. A. C., Ann. Rev. Biochemistry, (1946) 15, 1-34; Thompson, E. O. P., Biochim. Biophys. Acta, (1954) 15, 440; Stahman, M. A. and Spencer, A. K., Biopolymers, (1977) 16, 1299-1306). Such inadvertent reactions induce heterogeneity into the final reaction products.
Therefore, it would be desirable to use selective disulfide bond oxidants to generate antiviral peptides. As detailed below, the oxidant, potassium monopersulfate (E.I. Dupont de Nemours & Co., Wilmington, Del.), employed herein, is an alternate reagent that specifically oxidizes the sulfur-containing amino acids, cystine and cysteine, without the undesirable alterations cited above.
An alternate means for specific scission of disulfide bonds is through specific reductions, with subsequent alkylations of the resultant sulfhydryl groups with adducts that prevent reoxidation. Such reactions are commonly employed in determination of protein structure (Sela, M., White, F. H., and Anfinsen, C. B., Science, (1957) 691-692; Crestfield, A. M., Moore, S., and Stein, W. H., J. Biol. Chem., (1963) 238, 622). While reductions with sulfhydryl compounds such as dithiothreital and beta-mercaptoethanol are commonly employed, the phosphenes used herein represent alternative reductants for the same purpose of reducing the disulfide bonds in proteins (Ruegg, U. T. and Rudinger, J., Methods in Enzymol., (1977) 47, 111-126; Getz, E. B., Xiao, M., Chakrabarty, T., et al, Anal. Biochem. (1999) 273, 73-80).
The present invention also utilizes reduction reactions for generation of antiviral peptides that are viral neuraminidase inhibitors, procedures that likewise avoid inadvertent modifications of chemical groups required for full therapeutic effects. In addition, an important objective of the present invention is to obtain a subset of antiviral peptides utilizing reduction and alkylation based on selection, from a list of alkylating agents, of adducts that block the reoxidation of the disulfide bonds when the excess reductants are removed from the peptides. As detailed below, selections among those adducts can yield compounds that, through introduction of highly sensitive markers, permit (1) detailed studies of the interactions between the respective peptide and the virus preparations, and (2) modulation of interactions with the viral neuraminidases.
As detailed below, the antiviral peptides of the invention are useful for inhibiting viral neuraminidases and thus inhibiting viral infections. The myxoviruses are comprised of two families, the orthomyxoviruses and paramyxoviruses. Orthomyxoviruses include the Influenza viruses A, B, and C. Influenza A is associated with the most severe form of human influenza. It can infect a variety of mammals and birds. Birds and pigs can serve as reservoirs from which the virus can jump to humans, dogs, and other animals. One particularly prevalent subtype of Influenza A is the “avian flu”, subtypes generally denoted by HxNy. The “avian flu” is highly contagious and dangerous, especially with mutations that cannot be vaccinated against before such mutations have occurred. Paramyxoviruses 1 and 3 likewise cause flu-like symptoms but less severe than those of the Influenza A virus. Among other paramyxoviruses are the mumps virus, the Sendai virus (a mouse pathogen), and the Newcastle Disease virus, an important avian virus. On the surfaces of those viruses are two glycoproteins that contribute to viral infectivity and replication processes. One, a hemagglutinin, serves as the receptor-binding function for viral attachment to permissive cells. The other glycoprotein, a neuraminidase, facilitates release of newly formed virus particles that assemble in clusters inside the infected cells. The release is attributed to the hydrolytic cleavage of sialic acid residues by that enzyme. Thus, neuraminidase inhibitors are modulators of virus dissemination.
A clinical need for agents that block myxovirus production (e.g., influenza A virus) arises when the viruses undergo antigenic drift such that antibodies to a contemporary vaccine are rendered ineffective. Effective therapeutic compounds, zanamivir and oseltamivir, function well when administered early in the infectious process. Both compounds are analogues of sialic acid and function as inhibitors of the viral neuraminidase, thus inhibiting release of newly-generated varions from the infected cells, and blocking spread of the disease.
Historic evidence of a neurotropic component to the influenza A virus of the 1918 pandemic was suggested by reports of major CNS disturbances (Crookshank, F. G., Lancet, (1919) 1, 79-80). Neurotropic strains of influenza A virus were found to infect the brain stem via the olfactory bulb (Mori, I., Diehl, A. D., Chauhan, A., et al., J. Neurovirol. (1999) 5, 355-362), and axonal transport of the virus was clearly demonstrated (Matsuda, K., Shibata, T., Sakoda, Y., et al., J. Gen. Virol. (2005) 86, 1131-1139). The peptides described herein contain amino acid sequences common to components of surface proteins of some neurotropic viruses such as rabies viruses (Lentz, T. L., Hawrot, E., and Speicher, D. W., Science (1984) 226, 847-848: Lentz, T. L. Biochemistry, (1991) 30, 10949-57). Likewise, a glycoprotein constituent of the HIV virus displays an amino acid sequence homology with that of the rabies viruses and alpha-neurotoxins (Neri, P., Bracci, L., Rustici, M., et al., Arch. Virol. (1990) 114, 265-269: Bracci, L., Ballas, S. K., et al., Blood, (1997) 90, 3623-3628). The contribution of those structures to inhibition of myxovirus neuraminidases is suggested by peptide-virus complexes described herein.
In addition to treatment of human patients with myxovirus infections, an additional target for this invention is application of the products to treatments of a broad range of veterinary diseases. Recent evidence suggests that the influenza viruses can jump to dogs and other animals that may serve as viral reservoirs. Acute and chronic respiratory and/or gastrointestinal viruses are a constant problem in veterinary practice. Contagion expresses itself where dogs and cats are confined in animal hospitals, boarding facilities, pet stores, animal control facilities, zoos, etc. Viral infections affecting dogs include canine distemper, adenovirus type 2, corona virus, and the parainfluenza and parvoviruses. Among other infectious diseases affecting cats are rhinotracheitis, calici virus, panleukopenia and herpes viruses. Mortality rates in these infections are relatively high, especially in young animals. The diseases are spread by close contact with other animals or by contact with body discharges as result from coughing, sneezing, fecal contamination, etc. Other diseases are spread by more intimate contact such as by copulation, fighting, birthing, etc. Diseases acquired by those modes include feline leukemia (FeL) and infection with the feline immunodeficiency virus (FIV). Therefore, there is a need for antiviral peptides capable of treating or vaccinating against diseases affecting both humans and animals.